Mechanical strain plays a key role in bone homeostasis. Conversely, removal of mechanical strain (as in immobilization or weightlessness) has been shown to diminish or arrest bone formation, reduce collagen and bone matrix protein production, and decrease the mechanical properties of bone. The goal of this application is to determine the signaling mechanisms for mechanical strain and how mechanical unloading affects these mechanotransduction pathways. The group has previously characterized a nonselective cation, mechanosensitive channel (SA-cat) in the UMR-106.01 osteosarcomal cell line. They have demonstrated that this channel can be primed by activation of chronic intermittent strain and, using antisense oligodeoxynucleotide strategy, demonstrated that this channel appears to be an alternatively spliced isoform of the dihydropyridine-sensitive L-type calcium channel. They have further shown that the dihydropyridine-sensitive L-type channel is also involved in mechanotransduction in response to hypotonic swelling. The hypothesis to be tested in this application is that these channels act in the signal transduction mechanism for physical strain on bone, and that they may be down-regulated when mechanical strain is removed. Using chronic mechanical strain on osteoblasts in vitro, in conjunction with patch clamp analysis, they have designed experiments to study the effects of mechanical unloading on the SA-cat channels and L-type calcium channels. They will also investigate the interaction of these channels with the cytoskeleton and integrins of the osteoblast during mechanical unloading. The Specific Aims of this research are to: 1) ascertain changes in the SA-cat and L-type calcium channel in response to mechanical unloading; 2) determine the relationship of these channels to osteoblast proliferation and differentiation following mechanical loading and unloading; 3) correlate the effects of the cytoskeleton reorganization, following mechanical strain, with channel activity; and 4) determine the effects of integrin signaling on channel activity in osteoblast function. These studies are intended to provide new insight into the mechanism of transduction of the biophysical stimuli into osteoblast function.